The complex network of cytokines involved in inflammatory and immunoregulatory responses plays a substantial role in the pathogenesis of human immunodeficiency virus (HIV) infection. RANTES is a chemoattractant for CD4+/CD45RO T cells and is produced from various cell types including CD8+ T cells, CD4+ T cells and monocyte/macrophages (M/M). It has recently been shown to be capable of suppressing HIV replication in T cells. To better understand the molecular mechanisms of RANTES expression, the RANTES promoter region was analyzed by transient expression assays and gel-mobility shift assays. We have demonstrated that: (1) RANTES promoter activity was upregulated by PMA plus ionomycin, proinflammatory cytokines (tumor necrosis factor-alpha [TNF-alpha] and interleukin [IL]-1beta), or the CD28 costimulatory pathway; (2) the RANTES promoter region contains four NF-kappaB binding sites at positions -30, -44, -213, and -579 relative to the transcription start site; (3) one site (-213) is also an NF-ATp binding site, and the most distal site (-579) also serves as a CD28-response element; and (4) mutation of any of these NF-kappaB sites or co-expression of IkappaBalpha markedly reduces the promoter activity. These results indicate that NF-kappaB, a potent transcriptional activator of HIV expression, can also stimulate expression of RANTES, an anti-HIV chemokine.